IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. Regulation of RNA binding activity of IRP1 involves a transition from a form of IRP1 in which a 4Fe-4S cluster is bound, to a form that loses both iron and aconitase activity. The 4Fe-4S containing protein does not bind IREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to determine whether IRP1 will bind RNA. Over the past decade, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS, ISCU and Nif U, ferredoxin and ferredoxin reductase genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron- sulfur cluster of IRP1. We have discovered that frataxin transcription is iron-dependently regulated and frataxin expression decreases when there is cytosolic iron deficiency in wild-type and in fibroblasts and lymphoblasts from Friedreich ataxia patients. We discovered that a mutation in the scaffold protein, ISCU, causes a rare myopathy. In both Friedreich ataxia and ISCU myopathy, our data indicate that mitochondrial iron overload occurs in conjunction with cytosolic iron depletion. In collaboration, we discovered that mutations in NFU1 and BOLA3 mutations cause a human disease characterized by lactic acidosis and lipoic acid deficiency. We predicted that other rare genetic diseases characterized by mitochondrial compromise were caused by mutations in the genes responsible for iron-sulfur cluster biogenesis, and we collaborated to discover that mutations of NFS1 cause neonatal mitochondrial disease. We are characterizing the steps that chaperone transfer of nascent iron-sulfur clusters from their association with the initial assembly apparatus to proteins that require iron-sulfur clusters for function. We have extensively studied the metabolic remodeling of skeletal muscle metabolism in ISCU myopathy and discovered several compensatory pathways that help to maintain energy homeostasis. We have also discovered multiple reasons that limit the phenotype of ISU myopathy to skeletal muscles, while largely sparing other tissues. We are developing antisense treatment therapy for ISCU myopathy, and we recently demonstrated that FGF21 is a good biomarker for muscle disease in ISCU myopathy. We are also actively working to discover how SDHB acquires its three Fe-S clusters, and we have demonstrated that HSC20 cochaperone mediated iron sulfur cluster delivery is critical for iron sulfur acquisition of respiratory chains I-III. We are evaluating many more candidate recipients of iron sulfur clusters, and we expect our studies will greatly increase the number of known mammalian iron sulfur proteins. We established that Fe-S biogenesis occurs de novo in the cytosol, and that the chaperone HSC20 connects initial cytosolic biogenesis with the CIAO1-dependent Fe-S delivery platform in the cytosol by binding to a LYR motif in CIAO1. Thus Fe-S biogenesis occurs in parallel both in the mitochondrial matrix and in the cytosol of mammalian cells. Our work challenges the paradigm that initial Fe-S biogenesis occurs only in the mitochondrial matrix, and ABCB7 exports a component critical to Fe-S synthesis in mammalian cytosol. We are working to clarify the molecular interactions that promote transfer of Fe-S clusters to recipients using the HSC20 cochaperone system, followed in some cases by use of secondary scaffold proteins that confer specificity to subgroups of Fe-S recipients. Using informatics looking for iterations of the LYR motif, followed by overexpression and ICP-MS, we are in the process of identifying previously unrecognized Fe-S proteins, which we believe are common and represented in multiple key metabolic pathways of mammalian cells.